CN101201336A - Method for early evoking and identifying transformant of tobacco nicotine - Google Patents
Method for early evoking and identifying transformant of tobacco nicotine Download PDFInfo
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- CN101201336A CN101201336A CNA2006101283643A CN200610128364A CN101201336A CN 101201336 A CN101201336 A CN 101201336A CN A2006101283643 A CNA2006101283643 A CN A2006101283643A CN 200610128364 A CN200610128364 A CN 200610128364A CN 101201336 A CN101201336 A CN 101201336A
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Abstract
The invention discloses a method of early inducement identification of converter plants by tobacco smoke alkaloid. Plants are fixed and leaves are picked; any two kinds of or all of potassium bicarbonate, sodium bicarbonate and sodium chloride are mixed at the volume ratio of 1:1 or 1:1:1. The mixture is sprayed, air cured and dried. Nicotine and nornicotine are measured with a gas chromatograph; and the content of the nornicotine in the total quantity of the nicotine and the nornicotine is calculated. If the content of the nornicotine is less than 5 percent, the plants are not converter plants. If the percent conversion of the nicotine is larger than 5 percent, the plants are converter plants; and if the percent conversion of the nicotine is larger than 20 percent, the plants are high converter plants. The technical proposal of the invention has the following advantages: 1. the method adopted by the invention can ensure conversion to be completed within four days; the time is short, and the efficiency is high; 2. the chemical substances adopted by the invention are gentle, and have excellent effect; 3. the chemical substances adopted by the invention are environmentally friendly; 4. the cost of the chemical substances adopted by the invention is far below the cost of the processing method adopting ethephon.
Description
Technical field
The present invention relates to a kind of method for early evoking and identifying transformant of tobacco nicotine.
Background technology
Alkaloid is the important chemical constitution of a class that tobacco plant contains, and mainly comprises four kinds of nicotine, nornicotine, anatabine and anabasines.It all is the nicotine accumulation type that the common tobacco (N.tabacum L) and the makhorka (N.rustica L) of cultivation value are arranged, and its nicotine content accounts for more than 94% of total alkaloid content.But in the cigarette group of hill body of cultivar, indivedual plant can form nicotine demethyl ability because of gene mutation, cause nicotine content significantly to reduce, the corresponding increase of nornicotine content, and this have nicotine and be called transformant to the cigarette strain of nornicotine conversion capability.Because it is closely related that nicotine transforms with change of tobacco leaf fragrance and the increase of tobacco content of nitrosamines, become the focus of international academic community research in recent years.
(1) nicotine is to the influence of nornicotine conversion to quality of tobacco and TSNA
Nicotine transforms and causes tobacco leaf nornicotine content to raise unusually.Nornicotine is easy at the tobacco leaf modulated process and in the ageing process after modulating biochemical conversion takes place form many unfavorable compositions, as myosmine, acidylate nornicotine and N-nitrosonornicotine (NNN), thereby influences tobacco incense flavor quality and availability.
The influence of 1 pair of tobacco incense flavor quality
The appearance of nicotine transformant and accumulation cause tobacco leaf chemical composition to form the generation marked change in the tobacco colony.The oxidation of nornicotine can produce myosmine.Along with the increase of nornicotine content in the tobacco leaf, the blade and the myosmine content straight line in the master pulse of modulation back tobacco leaf increase.The acyl groupization of nornicotine forms the nornicotine derivant of a series of 1-8 of containing carbon atom acyl moieties.The increase of tobacco leaf nornicotine content causes the decline of fragrance quality.Roberts (1988) discovers that nornicotine produces myosmine and pyridine compounds in pyrolytic process, and flue gas is had such as peculiar smell such as alkali flavor, Mousiness.In cured tobacco leaf, the transformant tobacco leaf with higher nornicotine content shows " cherry is red " (cherry red) feature in appearance.This class tobacco leaf is because fragrance is relatively poor, thus when purchase row level separately, price is extremely low.Have the burley tobaccos TN90 strain of different nicotine conversion capabilities, economical character does not have significant difference, but the tobacco incense flavor quality increases significantly with nornicotine content and descend, and the general burley tobaccos style of transformant tobacco leaf descends, fragrance not just, the oral cavity is residual serious.
2 pairs of influences that tobacco leaf TSNA forms
NNN is a tobacco-specific nitrosamine (TSNA) main in tobacco leaf and the flue gas.Animal experiment shows that NNN has significant carcinogenic activity.Nitrosation reaction formation NNN very easily takes place in nornicotine in the modulated process of nicotine transformant.Shi etc. have selected 30 strains to have the cigarette strain (nornicotine content from 0.05% to 3.0%) of different nornicotine content, measure TSNA content in the tobacco leaf respectively, find nornicotine and NNN content significant positive correlation, and related coefficient is 0.9; Show also that with the dissimilar tobacco leaves mensuration of China the two has similar correlationship.In another test, difference by alkaloid and TSNA content in comparison transformant and non-transformant tobacco leaf and the master pulse, find that NNK, NAT and NAB do not have significant difference between transformant and non-transformant, increase considerably but NNN content increases with nornicotine content, also increase thereupon total NNN accounts for the TSNA content ratio.These results fully show and are transformed by nicotine that to cause nornicotine content to increase be the key factor that tobacco leaf NNN forms and accumulate, and the removal transformant is TSNA content and raising and improve the effective way of tobacco incense flavor quality in reduction tobacco leaf and the product from colony.
(2) probability and the period that nicotine transforms in the cultivation tobacco
Although in burley tobaccos, also find to have the problem of nicotine conversion as far back as the 1950's, but up to today this problem just draw attention, burley tobaccos modulation back is a dark-brown, so the tobacco leaf of transformant do not have notable feature in appearance, and this brings very big inconvenience to research.
Commercial burley tobaccos kind generally contains the transformant of 15-20% in colony, wherein the nicotine conversion rate of about half transformant is more than 20%.In the burley tobaccos TN90 kind that Shi Hongzhi etc. (2001) also find in China, contain 20% the transformant of having an appointment, but the nicotine transforming degree difference of different transformants, the nicotine conversion rate of high transformant can reach 80%.Discover that further China burley tobaccos master plants the nicotine transformant ratio of No. one, cenospecies Hubei Province cigarette up to more than 40%, this mainly is owing to contain due to a large amount of transformants as the B21 sterile line of female parent.
In the grown in field process, the nicotine with transformant of nicotine conversion capability transforms rare, and nicotine transforms preceding 3 week that mainly occurs in results back tobacco leaf modulated process, and flavescence changes less after latter stage.It is relevant that this death with the tobacco leaf cell causes the demethyl enzymatic activity to stop.
(3) necessity of transformant early evoking evaluation
In burley tobaccos, because non-transformant is higher to the transformant mutation rate, transformed gene is a dominant gene, lacks the visual discrimination method of outward appearance, and tobacco colony can accumulate a certain proportion of transformant with higher nornicotine content.
Transformant in the colony must be differentiated and be removed at the commitment of tobacco leaf growth (refer to tobacco leaf bloom pollination before), breed to leave and take pure non-transformant in assurance breeding of new variety and the stock breeding, or strict father, the maternal non-transformant selected hybridized the production cenospecies in hybrid seeding.Because non-transformant and transformant do not have difference on mode of appearance, and the nicotine of transformant transforms and mainly takes place in the tobacco leaf modulated process, so the early stage discriminating of transformant at first needs the nicotine conversion of transformant is stimulated, so that the early expression transformation traits.
(4) present Research
Shi Hong aims at the U.S. and is during the post-doctor, carries out the research of transformant evaluation aspect in early days in the world first, is to have adopted the ethephon (CEPHA) disposal route at first.Promptly before tobacco leaf is bloomed, win single leaf, tiling sprays the ethephon (CEPHA) aqueous solution, the drying in the air system of nature of preserving moisture then, treat the tobacco leaf flavescence after, measure the nicotine and the nornicotine content of tobacco leaf, calculating nicotine conversion rate.Though this method is effective, has following shortcoming: the one, the time is long.Because suitable temperature and concentration conditions are not studied, the system of drying in the air under field conditions (factors) generally needs the time more than 8 days that nicotine is fully transformed comprehensively.But need measure a large amount of samples in the limited time in actual applications, the time is extremely important.The 2nd, what ethephon (CEPHA) was handled induces effect relatively low, mainly be because ethephon (CEPHA) is the hormone releasing agent, to the concentration requirement strictness, be not easy accurate grasp, tend to occur owing to spray inhomogeneous, cause treatment effect inconsistent, cause the blade-section necrosis in the time of too much, low excessively, do not have effect again, therefore, usually can not identify the low low transformant of conversion capability.The 3rd, ethephon (CEPHA) is influential to environment, and pollution is arranged.The 4th, the cost height.
Summary of the invention
The purpose of this invention is to provide a kind of tobacco leaf transformant of inducing and carry out in early days the method that nicotine transforms to greatest extent, thereby provide condition for quality breeding, stock breeding, hybrid seeding in growth.
For achieving the above object, the technical solution used in the present invention is: a kind of method for early evoking and identifying transformant of tobacco nicotine may further comprise the steps:
A, picking leaves in tobacco leaf is transplanted 2～4 weeks of back, are plucked the 3rd or the 4th leaf from cigarette strain bottom number;
B, decide strain in the field, mark strain number is got behind 1 leaf on petiole in every strain;
C, spray, adopt saleratus, sodium bicarbonate, sodium chloride or any two or three equal-volume mixed solution wherein respectively, leave dual sides is evenly sprayed;
D, the blade string is hung after spraying, plastic covering cloth is preserved moisture, or the system of drying in the air in climatic chamber, dries when the rear blade flavescence changeed brown in 3～4 days;
E, employing gas chromatograph carry out nicotine and nornicotine mensuration, calculate the number percent that nornicotine content accounts for nicotine and nornicotine summation, if ratio is lower than 5%, are non-transformant; Nicotine conversion rate promptly is a transformant greater than 5%, wherein greater than 20% be high transformant.
The solution concentration that sprays use among the described step c is: saleratus and sodium chloride are 1.1～1.3%, and sodium bicarbonate is 0.9～1.1%; The climatic chamber temperature of using in the described steps d is controlled at 26～35 ℃, and relative humidity is not less than 65%.
Preferred scheme is that the solution concentration that sprays use among the described step c is: saleratus and sodium chloride are 1.2%, and sodium bicarbonate is 1.0%.
Too early, the cigarette strain was less for well 2～4 weeks after transplanting to processing time of tobacco leaf among the present invention, the sample size deficiency, and influence cigarette strain normal growth.The picking leaves position with from the bottom several 3-4 leaves be advisable, the middle and upper part leaf not fully the expansion blade should not pluck, the test show that underdeveloped transformant tobacco leaf can not fully transform after processing.The chemical substance concentration that adopts reduces, and can prolong transformation time, and can cause conversion not exclusively, reduces identification result.
The employing technical solutions according to the invention have the following advantages: 1, the method applied in the present invention can guarantee at 4 days with interior conversion, time weak point, the efficient height finished.2, chemical substance of the present invention is relatively gentle, and effect is better.3, chemical substance of the present invention is environmentally friendly.4, the cost of chemical substance of the present invention is far below the cost that adopts the ethephon (CEPHA) disposal route.
Embodiment
Under field condition, plant 120 strain TN90 high-nicotine transformant materials, transplant 3 weeks of back, respectively the strain sampling is decided in 120 cigarette strains, portion's the 3rd leaf is taken off in every strain, totally 120 leaves, it is divided into 6 groups, get 10 for every group and use 1.2% saleratus respectively, 1.0% sodium bicarbonate, 1.2% sodium chloride, the equivalent of these three kinds of solution (volume) mixed liquor, the equivalent of 1.2% saleratus and 1.0% sodium bicarbonate (volume) mixed liquor, equivalent (volume) mixed liquor of 1.0% sodium bicarbonate and 1.2% sodium chloride carry out that the blade face is two-sided to spray, in climatic chamber 28 degrees centigrade afterwards, under the 75% relative humidity condition, the system of drying in the air 4 days, oven dry then is with gas Chromatographic Determination nicotine and nornicotine content, calculate nicotine conversion rate, 6 groups of average nicotine conversion rates of handling are respectively: 92.5%, 93.2%, 90.3%, 91.6%, 93.8%, 90.4%, and the nicotine conversion rate of untreated contrast blade only is 28%.
For the evaluation of verifying early stage transformant with remove clean-up effect to the cigarette strain of offspring land for growing field crops colony, we adopt 1.2% saleratus and 1.0% sodium bicarbonate equivalent (volume) mixed liquor that burley tobaccos TN90 has been carried out the early stage evaluation of transformant, and have further studied the early stage performance of identifying the dissimilar cigarette strains in back the self progeny.According to the height of qualification result the cigarette strain is divided into non-transformant, hangs down transformant and high transformant by nicotine conversion rate, choose representational cigarette strain respectively and carry out selfing, next year, the self progeny to all types of cigarette strains planted, and the cigarette strain of each inbreeding population was carried out the evaluation of transformant.The result shows that the self progeny colony 100% of high transformant is high transformant, further specify the nicotine transformation traits and be the Gene Handling of showing one's color, and heredity is more stable; Segregation phenomenon appears in low transformant progeny population, and 28.5% high transformant is arranged approximately, and 31.3% low transformant and 40.2% non-transformant illustrate that the gene that control nicotine transforms is in heterozygous state; The progeny population of non-transformant has 91.4% cigarette strain still to be non-transformant, though the minority transformant occurred, but transforming degree is lower, early stage evaluation by transformant is described, strictness selects for use non-transformant to carry out seed production, to reducing progeny population nicotine transformant ratio, it is highly effective reducing the tobacco leaf nicotine conversion rate.
3 weeks after transplanting, choose 60 cigarette strains and carry out deciding strain, portion's the 3rd leaf is taken off in every strain, adopt 1.2% saleratus disposal route to carry out the evaluation of inducing of transformant, found that the nicotine conversion rate of 23 strain tobacco leaves reaches 5%, be the nicotine transformant, account for 38.3% of total mensuration strain number, wherein the nicotine conversion rate of 10 strains surpasses 20%, is high transformant, accounts for 16.7% of total mensuration strain number.After the tobacco leaf maturation, carry out the half whole strain system of drying in the air by normal mode, the sampling second time is carried out in the cigarette strain that the cured leaf phase is decided strain to the field, measures nicotine and nornicotine content, calculates nicotine conversion rate.The result shows have the nicotine conversion rate of 18 strain tobacco leaves to reach more than 5% in survey cigarette group of hill body.By twice measurement result more as can be seen, the transformant that identifies has in early days comprised all 18 transformants that form naturally after modulation, and distribution trend has higher consistance between the strain of the nicotine conversion rate of twice mensuration gained, illustrates by abductive approach and identifies that in early days transformant is highly effective.
The result also shows, early stage transformant induce nicotine conversion rate after the resulting nicotine conversion rate in back generally is higher than the tobacco natural system of drying in the air, the nicotine that further specifies processing such as saleratus and can stimulate and promote to have the strain of nicotine demethyl ability cigarette transforms to nornicotine, and this is very favourable to accuracy and the validity that improves transformant and identify.More meaningfully, can identify the low transformant that some can't effectively be differentiated after treatment under the system condition of normally drying in the air.In this test, there are 5 low transformants only just to be differentiated that therefore, the method can be differentiated low transformant effectively under the inductive condition in early days, thereby improve the effect of identifying.
Claims (3)
1. method for early evoking and identifying transformant of tobacco nicotine is characterized in that may further comprise the steps:
A, picking leaves in tobacco leaf is transplanted 2～4 weeks of back, are plucked the 3rd or the 4th leaf from cigarette strain bottom number;
B, decide strain in the field, mark strain number is got behind 1 leaf on petiole in every strain;
C, spray, adopt saleratus, sodium bicarbonate, sodium chloride or any two or three equal-volume mixed solution wherein respectively, leave dual sides is evenly sprayed;
D, the blade string is hung after spraying, plastic covering cloth is preserved moisture, or the system of drying in the air in climatic chamber, dries when the rear blade flavescence changeed brown in 3～4 days;
E, employing gas chromatograph carry out nicotine and nornicotine mensuration, calculate the number percent that nornicotine content accounts for nicotine and nornicotine summation, if ratio is lower than 5%, are non-transformant; Nicotine conversion rate promptly is a transformant greater than 5%, wherein greater than 20% be high transformant.
2. method for early evoking and identifying transformant of tobacco nicotine as claimed in claim 1, it is characterized in that the solution concentration that sprays use among the described step c is: saleratus and sodium chloride are 1.1～1.3%, and sodium bicarbonate is 0.9～1.1%; The climatic chamber temperature of using in the described steps d is controlled at 26～35 ℃, and relative humidity is not less than 65%.
3. method for early evoking and identifying transformant of tobacco nicotine as claimed in claim 1 or 2, it is characterized in that the solution concentration that sprays use among the described step c is: saleratus and sodium chloride are 1.2%, and sodium bicarbonate is 1.0%.
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| CNA2006101283643A CN101201336A (en) | 2006-12-12 | 2006-12-12 | Method for early evoking and identifying transformant of tobacco nicotine |
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| CNA2006101283643A CN101201336A (en) | 2006-12-12 | 2006-12-12 | Method for early evoking and identifying transformant of tobacco nicotine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949903A (en) * | 2010-09-09 | 2011-01-19 | 河南中烟工业有限责任公司 | Method for determining free nicotine in tobacco |
| CN105717110A (en) * | 2016-04-25 | 2016-06-29 | 安徽中烟工业有限责任公司 | Method for rapid identification on burley tobacco converter |
| CN111505158A (en) * | 2020-05-09 | 2020-08-07 | 云南省烟草农业科学研究院 | Method for rapidly screening chromaticness of nicotine-converted tobacco plants based on hatching treatment |
| CN111505157A (en) * | 2020-05-09 | 2020-08-07 | 云南省烟草农业科学研究院 | Non-hatching treatment-based method for rapidly screening chromaticness of nicotine-converted tobacco plants |
-
2006
- 2006-12-12 CN CNA2006101283643A patent/CN101201336A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101949903A (en) * | 2010-09-09 | 2011-01-19 | 河南中烟工业有限责任公司 | Method for determining free nicotine in tobacco |
| CN101949903B (en) * | 2010-09-09 | 2012-12-12 | 河南中烟工业有限责任公司 | Method for determining free nicotine in tobacco |
| CN105717110A (en) * | 2016-04-25 | 2016-06-29 | 安徽中烟工业有限责任公司 | Method for rapid identification on burley tobacco converter |
| CN105717110B (en) * | 2016-04-25 | 2018-05-01 | 安徽中烟工业有限责任公司 | A kind of method of burley tobaccos transformant Rapid identification |
| CN111505158A (en) * | 2020-05-09 | 2020-08-07 | 云南省烟草农业科学研究院 | Method for rapidly screening chromaticness of nicotine-converted tobacco plants based on hatching treatment |
| CN111505157A (en) * | 2020-05-09 | 2020-08-07 | 云南省烟草农业科学研究院 | Non-hatching treatment-based method for rapidly screening chromaticness of nicotine-converted tobacco plants |
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Open date: 20080618 |